Oligos for NGS

I performed PCR from SAM library and NGS to determined sgRNA distribution. I ordered the oligos for the PCR from Macrogen (purification MOPC). NGS results showed a passing filter of 96% but with a quality of the index read low and we had to allow one mismatch in the index reads. I was wondering if this problem was related with my oligos. Do you think that I should order oligos as 4-nmol ultramers as Julia recommend in her paper or it will not help. Has anybody used oligo primers, instead of ultramers, for this type of PCR?


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Fuchsia Kelpieabout 2 years ago

The quality (% > Q30) and cluster passing filter can be independent. Is the 96% cluster passing filter? If so, then the low quality of the index read may be a result of overloading the sequencer or not providing enough diversity.

It's also possible this is related to the oligo quality. From IDT, we have noticed that if we order long oligos at 1umol synthesis scale, rather than ultramers, the oligos are much lower quality.

Sienna Pixieabout 2 years ago

First of all, I am very sorry for the delay.

Yes, it is 96% cluster passing filter. I will check what you said.

Thank you very much

Sienna Pixieabout 2 years ago

I am sorry to bother you again with the same problem. I double checked my oligos and I realized that I ordered oligos at 50nmol scale. You said 1umol synthesis scale. Am I doing something wrong? Do we need so much oligo?

Fuchsia Kelpieabout 2 years ago

To clarify, I was recommending ordering 4nmol ultramer oligos from IDT that have higher fidelity than the 1umol synthesis scale. Since you are using a different oligo synthesis vendor, I can't say for sure if your oligos might be the problem. If you see this same problem in subsequent NGS runs, I would recommend ordering new oligos.

Sienna Pixieabout 2 years ago

Thank you very much!

I really appreciate your help.

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